Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs

Abstract

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50) values ranging from 20-100 µg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99) values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99) value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis.

Figure 1. Comparative homology modeling based three-dimensional structure of MtTPS.

(a) The overall structure of MtTPS showing the characteristic triosephosphate isomerase fold. The binding site groove of MtTPS (b) and PfTPS (c) is shown and the depth of the binding site groove is encircled. The structure is shown from a side view rotated 90 degrees along the vertical axis with respect to the top view. The figures were prepared by using Pymol Molecular viewer .

Figure 2. Verification of the docking procedures of Autodock4 and DOCK6.

(a) A Ribbon representation of MtTPS structure is shown in a side view rotated 90 degrees along the vertical axis with respect to the top view. The grid used for the docking of ligands is shown in a box representation. (b) The ligand CF₃HMP-PP bound at the active site of BsTPS in the X-ray crystal structure (PDB ID- 1G4P). Ligand docked by Autodock4 (c) and DOCK6 (d) at the active site of MtTPS. The ligand is shown in orange stick model. Figure (a) was prepared by software Autodock4 while the rest of the figures were prepared by using Pymol Molecular viewer .

Figure 3. Evaluation of the compounds for their potential to inhibit the activity of MtTPS.

Bar diagram represents percent inhibition of MtTPS activity in the presence of compounds at a concentration of 100 µg/ml. The data depicts the values as mean ± S.E. of two separate experiments carried out in duplicates.

Figure 4. Effect of compounds on the viability of M.smegmatis and M.tuberculosis.

The data depicts the values as mean ± S.E. of two separate experiments.

Figure 5. Evaluation of compound 9 for its cytotoxic effect on various cell lines.

Cell viability of THP-1, HeLa, HepG2 and HuH cells in the presence of varying concentrations of compound 9.

Figure 6. Differences in the binding site of MtTPS.

(a) Comparison of the residues at the binding site groove. The structures of MtTPS and PfTPS are superimposed (wheat colour), CF₃HMP-PP is bound at the binding site (yellow colour), residues Cys¹³⁹, Phe¹⁷⁴ and Arg¹⁹⁴ of MtTPS (red) and corresponding Gly¹²⁵ ,Val¹⁵⁸, Gly¹⁷⁸ of PfTPS (blue) are shown in stick model. (b) Molecular surface representation of the mutated MtTPS structure. In-silico substitutions of Cys¹³⁹, Phe¹⁷⁴ and Arg¹⁹⁴ in the MtTPS structure by Gly, Val and Gly resulted in a much deeper active-site groove than in the original structure. (c) Molecular surface representation of the original MtTPS structure. The depth of the groove is encircled. The figures were prepared by using Pymol Molecular viewer .

Figure 7. Interaction of compound 9 with the active site of MtTPS and its mode of action.

(a) Binding mode of compound 9 (NSC33472) at the active site of MtTPS. The figure was prepared by using Pymol Molecular viewer . (b) Inhibition of MtTPS activity by compound 9 in the presence of varying concentrations of HMP-PP.